Taxol, a diterpenoid extracted from tissues of the slow growing Taxus brevifolia Pacific yew) tree, shows considerable promise as an anticancer drug. However, a reliable large scale supply of taxol is not available. Tissue culture of Taxus may provide an alternative supply. Unfortunately, taxol, by binding to microtubules, is likely toxic to most cells; thus the development of a microculture system for the high level production of taxol may require the differentiation of cell types capable of partitioning the toxic compound away from actively dividing cells. To achieve this, we propose to develop a nodule culture system for Taxus based on work done with other woody plants. Nodules are dense cell clusters which form, grow and multiply as a cohesive unit. Nodules are particularly attractive for taxol production as they display a high degree of both cellular and tissue differentiation. Nodule cultures can theoretically serve as a production system for taxol since the outer rapidly dividing cell layers maintain the growth needed for biomass proliferation while the inner differentiated cells provide an isolated location for taxol production. Nodule cultures will also serve as a manipulatable model for studies of taxol biosynthesis. We will investigate the conditions which affect nodule development, growth and tissue HPLC taxane profiles. To understand the requirements taxol production, immunocytological localization of taxanes within intact plants and the seasonal environmental influences will be determined. Such information will be used to deduce the conditions required for maximal taxol synthesis in nodule cultures. The influence of batch and perfused bioreactor systems on production will also be determined. Once the development of a Taxus nodule system for enhanced taxol production is accomplished, then it can be used to extend the research to the elucidation of the enzymes and genes involved in taxol synthesis.